Cellular detection of infections [Elektronische Ressource] : analysis of systemic toll-like receptor 2 function and species specificity / Alina Grabiec

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Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2005
• Nombre de lectures: 20
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

Institut für Medizinische Mikrobiologie, Immunologie und Hygiene
der Technischen Universität München




Cellular detection of infections:
Analysis of systemic toll-like receptor 2 function
and species specificity


Alina Grabiec




Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt der Technischen Universität München zur
Erlangung des akademischen Grades eines

Doktors der Naturwissenschaften (Dr. rer. nat.)

genehmigten Dissertation.


Vorsitzender: Univ.-Prof. Dr. Rudi F. Vogel
Prüfer der Dissertation: 1. Univ.-Prof. Dr. Siegfried Scherer
2. Priv.-Doz. Dr. Stefan Bauer


Die Dissertation wurde am 20.10.2004 bei der Technischen Universität München
eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt am 22.12.2004 angenommen.


Table of contents

Table of contents
TABLE OF CONTENTS..............................................................................................1
LIST OF FIGURES......................................................................................................4
LIST OF TABLES .......................................................................................................5
ABBREVIATION LIST ................................................................................................6
1. SUMMARY............................................................................................................11
2. INTRODUCTION...................................................................................................13
2.1 Immune system ...................................................................................................................... 13
2.1.1 Innate Immunity .............................................................................................................. 13
2.1.2 Sepsis ............................................................................................................................. 14
2.2 Toll like receptors are pattern recognition receptors......................................................... 16
2.2.1 Drosophila Toll receptor and mammalian Toll Like Receptors (TLRs)........................... 16
2.2.2 TLR signaling.................................................................................................................. 18
2.2.3 TLRs mediate species specific PAMPs recognition ....................................................... 22
2.3 TLR2......................................................................................................................................... 24
2.3.1 TLR2 structure ................................................................................................................ 24
2.3.2 TLR2 function 26
2.3.3 TLR2 signaling 27
2.3.4 TLR2 and in vivo studies ................................................................................................ 27
3. AIMS .....................................................................................................................30
3.1 Identification of species-specific TLR2 agonist, as well as analysis of molecular
requirements .......................................................................................................................... 30
3.2 Analysis of role of TLR2 in the host response to Listeria monocytogenes infection in
vivo .......................................................................................................................................... 30
3.3 Analysis of potential interaction between TLR2 and specific cytoplasmic proteins...... 31
4. MATERIAL AND METHODS ................................................................................32
4.1 Materials.................................................................................................................................. 32


- 1 -
Table of contents

4.1.1 Reagents......................................................................................................................... 32
4.1.2 Buffers and solutions....................................................................................................... 32
4.1.3 KIT-systems .................................................................................................................... 36
4.1.4 Media............................................................................................................................... 36
4.1.5 Antibodies and antibody conjugates ............................................................................... 38
4.1.6 Plasmids.......................................................................................................................... 39
4.1.7 Oligonucleotides.............................................................................................................. 40
4.1.8 Bacterial strains............................................................................................................... 43
4.1.9 Cell lines 44
4.1.10 Mice.. 44
4.2 Methods ................................................................................................................................... 45
4.2.1 Site directed mutagenesis............................................................................................... 45
4.2.2 Splice PCR and point mutagenesis ................................................................................ 46
4.2.3 Restriction digest of DNA and ligation ............................................................................ 48
4.2.4 Transformation of E. coli ................................................................................................. 48
4.2.5 DNA Plasmid preparation in E. coli................................................................................. 49
4.2.6 Preparation of glycerol stocks......................................................................................... 49
4.2.7 Cell culture ...................................................................................................................... 49
4.2.8 Transfection of HEK 293 cells 50
4.2.9 Luciferase reporter assay................................................................................................ 51
-/-4.2.10 Electroporation of MEF TLR2 ....................................................................................... 52
4.2.11 Protein isolation and Western blot analysis .................................................................... 52
4.2.12 Immunodetection of protein ............................................................................................ 53
4.2.13 FACS analysis................................................................................................................. 54
4.2.14 ELISA - Enzyme Linked Immune Sorbent Assay ........................................................... 55
4.2.15 Immunocytochemistry ..................................................................................................... 55
4.2.16 EMSA - Electro Mobility Shift Assay ............................................................................... 55
4.2.17 Immunoprecipitation........................................................................................................ 57
4.2.18 Deglycosylation assay 58
4.2.19 Bacterial preparation....................................................................................................... 58
4.2.20 In vivo experiments and preparation of organs............................................................... 58
5. RESULTS AND DISCUSSION – PART I ............................................................. 60
5.1 Results..................................................................................................................................... 61
5.1.1 Comparative mutagenesis of wild-type h- and m TLR2.................................................. 61
5.1.2 Comparative analysis of TLR2-construct activities by genetic complementation of
HEK293 cells................................................................................................................... 62


- 2 -
Table of contents

-/-5.1.3 Comparative analysis of TLR2-construct activities by genetic complementation of TLR2
MEFs............................................................................................................................... 68
5.1.4 Immunocytochemical analysis of lipopeptide uptake by RAW 264.7 (mouse) cell line,
-/-THP-1 (human) cell line and macrophages from TLR2 mice ....................................... 69
5.1.5 Specific N-glycosylation of human and mouse TLR2..................................................... 71
5.2 Discussion .............................................................................................................................. 73
6. RESULTS AND DISCUSSION - PART II..............................................................75
6.1 Results...... 75
6.1.1 The survival of TLR2-deficient mice after infection with Listeria monocytogenes (LM).75
6.1.2 Bacteria load in TLR2-deficient mice upon infection with Lister